Difference between revisions of "Part:BBa K1965045"

 
 
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<partinfo>BBa_K1965045 short</partinfo>
 
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<p>This construct consists of the C-terminal part of the split luciferase attached to the coiled coil A. The sequence for the coiled coil A was taken from the Shekhawat <i>et al.</i> (<ref>1</ref>).  The construct also features a HA protein tag on the C-terminus. Coiled coil A dimerizes with the coiled coil B, which was also described in Shekhawat <i>et al.</i>.  If the B coil is fused to the N-terminal part of the split luciferase (<a href="https://parts.igem.org/Part:BBa_K1965046">BBa_K1965046</a>), the luciferase can reassemble and regain its activity through the dimerization of the coiled coil pair A and B (<ref>2</ref>A) <sup>[1]</sup> . </p>
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          <figcaption><b> Sequence alignment of different coiled coils used to tune the affinity of antiparallel coiled-coils.</b><br/> We designed different destabilized coils from our coiled coil pair AP4 and P3; Ile and Leu were substituted with Ala at the a and/or d position of the first and/or second heptad of P3mS (a more soluble variant of the original P3).</figcaption>
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          <figcaption><b> Interactions and protease activated AB coiled-coil formation.</b><br/> HEK293T cells were transfected with plasmids, coding for the appropriate coiled coil constructs. 24 h after transfection cells were lysed and luciferase activity was determined with the dual luciferase assay. (A) A and B coiled-coils, fused the split firefly luciferase spontaneously interact and reconstitute firefly luciferase. (B) A’:TEVs:B:nLuc and cLuc:A:PPVs:B’2A auto-inhibitory coiled coils reconstitute activity of firefly luciferase upon cleavage by TEV and PPV proteases. Successive luciferase reconstitution is observed only at high amounts of plasmids coding for the A’:TEVs:B:nLuc and cLuc:A:PPVs:B’2A constructs. </figcaption>
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<sup>[1]</sup> Shekhawat, S. S., Porter, J. R., Sriprasad, A., & Ghosh, I. (2009). An Autoinhibited Coiled-Coil Design Strategy for Split-Protein Protease Sensors. Jacs, 131(42), 15284–15290. <br>
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<!-- Add more about the biology of this part here
 
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Latest revision as of 17:32, 18 October 2016


cLuc:A:HA

This construct consists of the C-terminal part of the split luciferase attached to the coiled coil A. The sequence for the coiled coil A was taken from the Shekhawat et al. (1). The construct also features a HA protein tag on the C-terminus. Coiled coil A dimerizes with the coiled coil B, which was also described in Shekhawat et al.. If the B coil is fused to the N-terminal part of the split luciferase (BBa_K1965046), the luciferase can reassemble and regain its activity through the dimerization of the coiled coil pair A and B (2A) [1] .

Sequence alignment of different coiled coils used to tune the affinity of antiparallel coiled-coils.
We designed different destabilized coils from our coiled coil pair AP4 and P3; Ile and Leu were substituted with Ala at the a and/or d position of the first and/or second heptad of P3mS (a more soluble variant of the original P3).
Interactions and protease activated AB coiled-coil formation.
HEK293T cells were transfected with plasmids, coding for the appropriate coiled coil constructs. 24 h after transfection cells were lysed and luciferase activity was determined with the dual luciferase assay. (A) A and B coiled-coils, fused the split firefly luciferase spontaneously interact and reconstitute firefly luciferase. (B) A’:TEVs:B:nLuc and cLuc:A:PPVs:B’2A auto-inhibitory coiled coils reconstitute activity of firefly luciferase upon cleavage by TEV and PPV proteases. Successive luciferase reconstitution is observed only at high amounts of plasmids coding for the A’:TEVs:B:nLuc and cLuc:A:PPVs:B’2A constructs.
[1] Shekhawat, S. S., Porter, J. R., Sriprasad, A., & Ghosh, I. (2009). An Autoinhibited Coiled-Coil Design Strategy for Split-Protein Protease Sensors. Jacs, 131(42), 15284–15290.



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]